Hexavalent chromium[Cr(VI)]exists at the Superfund sites where it poses major health concerns to humans. Bacteria-mediated in situ transformation of water soluble and toxic Cr(VI) to less soluble/toxic Cr(III) represents a promising method to contain and treat Cr(VI). However, the molecular mechanisms by which bacteria reduce Cr(VI) remain undetermined. Reduction of Cr(VI) by dissimilatory iron (Fe) reducing bacterium Shewanella oneidensis results in formation of nano-sized Cr(III) particles on the cell surfaces. We hypothesize that cell surface-exposed cytochromes MtrC and OmcA of S. oneidensis are involved in the formation of Cr(III) by reducing Cr(VI) directly as Cr(VI) terminal reductases and indirectly as Fe(III) oxide terminal reductases to produce Fe(II) that in turn reduces Cr(VI). We plan to use nanoscale AFM correlated SERS imaging microscopy (AFM-CSIM) developed by us along with other methods to test our hypothesis. AFM-CSIM can simultaneously determine the locations of cytochromes and nano-sized metal particles as well as their oxidation states on the bacterial surfaces at nanometer-resolution. Co-localization of MtrC and OmcA with nano-sized Cr(III) and Fe(III) particles by AFM-CSIM will provide direct evidences to support our hypothesis. Other approaches (e.g. immuno-gold localization with scanning electron microscopy and the measurements of Cr(VI) reductase activity of purified MtrC and OmcA) will be used to validate the AFM-CSIM results. Successful completion of this project will help not only achieve our long-term goal of understanding the molecular mechanisms by which bacteria reduce Cr(VI), but also develop science-based solutions to mitigate the toxic effects of Cr(VI) at the Superfund sites.